Comprehensive HIV Protease Assay Protocol

More details on experimental assays, plasmid constructs, sequences, cell lines, antibodies and computational analysis are provided in Supplementary Methods. Briefly, affinity tagging and purification was carried out as previously described^{2 (link)} and the protein samples were analysed on a Thermo Scientific LTQ Orbitrap XL mass spectrometer. For the evolutionary analysis, genome-wide alignments to rhesus macaque were downloaded from the University of California, Santa Cruz genome browser (http://genome.ucsc.edu/) and evolutionary rates for each group of genes considered were measured using the synonymous and non-synonymous rates of evolution. For the in vitro protease assay, maltose binding protein (MBP)-tagged PR was expressed in BL21 (Gold) DE3 cells in the presence of 100 μM Saquinavir and purified on an MBP trap column. Purified eIF3 was obtained from J. Cate (University of California, Berkeley). For the infection assays, HeLa P4.R5 cells were transfected with short interfering RNAs and after 48 h infected with pNL4-3 or a pNL4-3-derived VSV-G-pseudotyped reporter virus. Infection levels were determined by luminescence read-out.